Apoptosis and autophagy in involuting bovine mammary gland is accompanied by up-regulation of TGF-beta1 and suppression of somatotropic pathway

Regulation of mammary gland remodeling during the lactation cycle in cattle still remains unclear. The present study focused on the role of TGF-beta1 and somatotropic pathways proteins in control of the switch between survival and death of bovine mammary epithelial cells (MEC). Expressions of TGF-beta1, TGF-betaRII, IGF-IRalpha, IGF-IRbeta, GH-R, IGFBP-3, -4, and -5 in mammary tissue explants in Holstein-Fresian heifers (n = 7) and cows (n = 23) in early lactation (1-100 day), late lactation (200-260 day) and drying off (280-340 day) were compared with biochemical indices of apoptosis (caspase 3, 89 kDa fragment of PARP) and autophagy (Beclin1). The results revealed that an increase in apoptosis during the dry period was accompanied by highly significant increases in TGF-beta1 and TGF-betaRII expression. Beside biochemical markers, typical morphological features of apoptosis, such as cell shrinkage, separation from the neighboring cells and condensation of chromatin were observed. TGF-beta1 expression and induction of apoptosis was facilitated by the suppression of somatotropic pathway during drying off, manifested with down-regulation of GH-R and IGF-IRalpha, and up-regulation of IGFBP-4 and -5. This is the first report describing autophagy in the bovine mammary gland. Similarly to apoptosis, the intensity of autophagy was the highest in the dry period, as shown by increased expression of Beclin1 and morphological features, e.g. autophagosomes, autophagic vacuoles. Autophagy observed in the involuting mammary tissue could be the natural cell defense against transient undernourishment and action of apoptogenic peptides (e.g. TGF-beta1, IGFBPs), thus maintaining cellular homeostasis in the dry period.     

Blastocyst development on days 10 or 14 after consumption of zearalenone by sows on days 7 to 10 after breeding

Sixteen primiparous sows were bred and fed either a control ration (n = 8) or a diet containing purified zearalenone (n = 8; 1 mg/kg of body weight) from days 7 to 10 after breeding. On day 7 after breeding, the jugular vein of each sow was cannulated and blood was collected at 20-minute intervals for 4 hours before feeding and 4 hours after feeding. On day 10 after breeding, blood samples were collected from 4 control sows and 4 zearalenone-fed sows at 20-minute intervals for 4 hours before collection of blastocysts. A similar blood sampling schedule was followed for the remaining 4 control and 4 zearalenone sows on day 14 after breeding. On day 10 after breeding, spherical blastocysts were recovered from all control sows and from 3 of 4 zearalenone-treated sows. Average diameter of blastocysts from zearalenone-treated sows were similar to that of control sows. On day 14 after breeding, blastocysts were recovered from all control sows and 3 of 4 zearalenone-treated sows. Blastocysts from the control sows were filamentous, whereas blastocysts from zearalenone-treated sows were fragmented and contained foci of necrosis. Incidence of luteinizing hormone (LH) secretory spikes per sow was less (P less than 0.01) in zearalenone-treated sows (0.25 +/- 0.25/4 h) than control sows (1.75 +/- 0.25/4 h) on day 10 after breeding. Incidence of follicle-stimulating hormone (FSH) secretory spikes was similar (P = 0.45) among treatments on days 7, 10, and 14 after breeding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of epidermal growth factor on growth of bovine mammary tissue in athymic nude mice

Bovine mammary tissue obtained from midpregnant Holstein heifers by surgical biopsy was transplanted subcutaneously to ovariectomized athymic nude mice (n = 5 heifers). After 3 weeks recovery, mice were either sham operated or sialoadenectomized (submandibular salivary glands removed). After an additional week, mice were injected with saline or 17 beta-estradiol + progesterone (1 microgram + 1 mg/day) for 2 days. In addition, half of the sialoadenectomized mice were injected with epidermal growth factor (5 micrograms/day). Grafted tissue was removed and rate of deoxyribonucleic acid (DNA) synthesis estimated by incorporation of 3H thymidine. Estradiol + progesterone increased the incorporation of 3H thymidine from 77 +/- 20 dpm/micrograms DNA to 472 +/- 53 dpm/micrograms DNA. In sialoadenectomized mice, DNA synthesis was increased from 88 +/- 16 dpm/micrograms DNA (saline treated) to 360 +/- 29 dpm/micrograms DNA (estradiol + progesterone treated). In sialoadenectomized mice treated with epidermal growth factor, DNA synthesis in estradiol + progesterone treated mice was 529 +/- 36 dpm/micrograms DNA, compared to 112 +/- 30 dpm/micrograms DNA in sialoadenectomized mice treated with epidermal growth factor. These data indicate that sialoadenectomy of athymic nude mice decreased the ability of transplanted bovine mammary tissue to increase DNA synthesis in response to estradiol and progesterone. This inhibition was removed by epidermal growth factor treatment. These data suggest a physiological role of epidermal growth factor in regulating development and hormone responsiveness of bovine mammary tissue.     

Cloning,expression, and tissue distribution of bovine interleukin-21

Bovine interleukin-21 (IL-21) cDNA was cloned and sequenced from bovine peripheral blood lymphocytes (PBLs) stimulated with 10 microg/ml concanavalin A (ConA), 10 microg/ml phytohemagglutinin (PHA), and 50 ng/ml phorbol 12-myristate 13-acetate (PMA) for 48 h. The open reading frame of the bovine IL-21 cDNA is 459 bp in length and encodes 152 amino acids. The predicted amino acid sequence is 78.2 and 58.5% homologous to the human and murine IL-21 amino acid sequences, respectively. Recombinant bovine IL-21 was expressed by a baculovirus expression system. The bovine IL-21 was processed to the mature form in insect cells and secreted to the supernatant confirmed by N-terminal amino acid sequencing. The recombinant bovine mature IL-21 induced the proliferation of human IL-2-dependent cells, ILT-MAT. The mRNA expression for bovine IL-21 was observed in the spleen, but not in the brain, heart, lung, liver, and kidney. The bovine IL-21 identified in this study may provide new methods for the enhancement of innate immunity in cows.     

Accelerated bovine mammary involution induced by infusion of concanavalin A or phytohemagglutinin

Right mammary quarters of dairy cows were infused with concanavalin A (conA) or phytohemagglutinin (PHA) near drying off. Significant changes in milk yield and composition were observed by 24 hours after infusion. In general, conA or PHA treatment resulted in significantly greater compositional changes earlier in the dry period compared with control quarters. As involution progressed, lactoferrin, serum albumin, immunoglobulin G, pH, and numbers of somatic cells increased, whereas citrate and the molar ratio of citrate:lactoferrin decreased. Mammary secretion accumulation at 7 days of involution was significantly lower in treated compared with control quarters. The data indicated that intramammary infusion of conA or PHA near drying off accelerates bovine mammary involution, resulting in elevated levels of natural protective factors.     

Distribution of immunoglobulin-bearing leukocytes in bovine mammary tissue infected chronically with Staphylococcus aureus

Mammary parenchymal and test end tissues from cows with chronic Staphylococcus aureus mastitis were examined to determine the distribution of immunoglobulin (Ig) G1- and IgG2-bearing leukocytes. Leukocytes bearing IgG2 predominated in S. aureus infected quarters, with highest numbers observed at the Furstenberg's rosette followed by streak canal and parenchymal tissue areas. Significantly more IgG1- and IgG2-bearing leukocytes were observed at the Furstenberg's rosette and significantly more IgG2-bearing leukocytes were observed at the streak canal of S. aureus infected quarters compared to uninfected quarters. Receptors for cytophilic IgG on neutrophils and macrophages may increase efficiency of phagocytosis and improve the antimicrobial effectiveness of these cells in treat end tissues.     

Adherence and colonization by bacterial pathogens in explant cultures of bovine mammary tissue

Explant cultures of bovine mammary tissue taken from virgin heifers were used to examine adherence, colonization and cytopathogenesis ofStreptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, Staphylococcus aureus andEscherichia coli in the putative target tissue. None of the five bacteria was able to adhere to healthy ductular epithelium but all showed a marked tropism for exposed connective tissue.S. aureus andE. coli induced a marked cytopathic effect in ductular epithelium after 6 hours in culture but the bacteria were not in close association with the affected tissue. No evidence could be found to support the hypothesis that adherence to epithelium might be the first stage in the pathogenesis of mastitis caused by these organisms.     

饲粮代谢能水平对22～42日龄黄羽肉鸡生长性能、胴体品质以及部分血液生化指标影响的研究

试验以岭南黄鸡为代表,研究饲粮代谢能水平对22～42日龄黄羽肉鸡生长性能、胴体品质以及部分血液生化指标的影响。试验结果表明:①随饲粮代谢能升高,试鸡平均日增重有增加趋势,平均日采食量有降低趋势,而饲料转化率得到极显著改善(P<0.01);②饲粮代谢能水平对试鸡胴体品质有显著(0.010.05)。     

Effect of teat number, teat abnormalities and underline length on litter sizes and weights at 21 and 42 days in swine

Data from 80 first-litter purebred and crossbred gilts of Duroc and Landrace breeding were analyzed to determine the effects of traits associated with their udder on the sizes and weights of their litters at 21 and 42 d. Traits measured on each first-litter gilt included: number of teats in front of the navel; number of functional teats; number of inverted and pin nipples; length of the underline; body length and weight at parturition. When the litter size nursed at 1 d was held constant, litter size at 21 and 42 d was not influenced by number of teats in front of the navel, number of functional teats, number of inverted nipples, number of pin nipples, body length or weight at parturition. First litter gilts with shorter underlines tended to raise larger litters at 21 and 42 d (P less than .05). Litter weights at 21 and 42 d were not affected by the number of teats in front of the navel, number of functional teats, number of inverted nipples, or by the number of pin nipples when the size and weight of the litters born alive were held constant. First-litter gilts with shorter body lengths tended to have heavier litters at 21 d, (P less than .05) while those with shorter underlines (P less than .05) and those that weighed more at parturition (P less than .10) tended to have heavier litters at 42 d. Results from this study are interpreted to indicate a replacement-gilt selection program that applies selection pressure for only increased teat number and against teat abnormalities seems unwarranted.     

Immunohistochemical localization of IgG1 and IgG2 in prepartum and lactating bovine mammary tissue

The differential distributions of IgG1 and IgG2 were determined in prepartum and lactating bovine mammary tissue by indirect immunofluorescence. IgG1 was found predominately within the alveolar epithelial cells and lumens of prepartum tissue whereas IgG2 was largely confined to the stromal area surrounding the alveoli. Both IgG subclasses were confined predominately to the stroma in lactating tissue. Few IgG containing stromal cells were readily distinguished in any of the mammary tissue used in this study.     

Changes in plasma progesterone concentrations from days 17 to 42 of gestation in mares maintaining or losing pregnancy

Plasma progesterone concentrations were measured in 179 mares bled on alternate days commencing with a positive pregnancy diagnosis on Days 17 to 18 after ovulation and concluding on Days 42 to 45. During this period 17 mares (10 per cent) lost their pregnancies, 11 before Day 25. In 15 mares the timing of the pregnancy loss could be determined with adequate accuracy; in only one did a decline in progesterone precede the loss. Thus pregnancy loss between Days 17 and 42 was rarely caused by a fall in plasma progesterone.     

Accuracy of bovine pregnancy detection using transrectal ultrasonography at 28 to 35 days after insemination

OBJECTIVE: To estimate the accuracy of real-time ultrasonography to detect pregnancy in dairy cows at 28 to 35 days after insemination. METHODS: Cows that did not return to oestrus between 18 and 24 days after a first insemination (n = 526) were examined by transrectal ultrasonography from 28 to 35 days after insemination. Pregnancy was confirmed by the observation of a foetus, but fluid in the uterine horn and the presence of embryonic membranes were also noted. When pregnancy was not confirmed by the observation of a foetus, a second examination 7 days later, confirmed these remaining cows as pregnant or not pregnant to the first insemination. Detection of pregnancy at this early examination was compared with manual transrectal pregnancy examination performed 10 to 13 weeks after insemination (13-week examination). RESULTS: There were 44% of cows that were pregnant to the first service, 34% that had returned for a second service 18 to 24 days after the first insemination, and 20% of cows that were not pregnant, and had not returned normally for a second service (non-pregnant, non-return) within 24 days of their initial insemination. The presence of a foetus at 28 to 35 days after insemination was accurately predicted by a simplified method where uterine fluid accumulation and embryonic membranes were observed. Foetal loss between the early detection and the 13-week examination (9% of pregnancies) indicated that 28 to 35 days post insemination was too early to reliably detect pregnancy. CONCLUSION: Early examination of pregnancy with transrectal ultrasonography is an accurate method to identify non-pregnant, non-return cows. The examination can be simplified by the observation of uterine fluid accumulation and embryonic membranes, as opposed to the more involved process of observing the foetus.     

The effect of lipopolysaccharide on bovine mammary macrophage function

The effect of Escherichia coli lipopolysaccharide (LPS) on the expression of major histocompatibility complex (MHC) class II molecules by bovine mammary macrophages was examined. The ability of LPS-treated mammary macrophages to support antigen-specific T-cell proliferation, as a measure of their antigen presentation ability, was also evaluated. For this purpose, control and LPS-treated macrophages were pulsed with heat-killed Staphylococcus aureus and then cultured with S. aureus-sensitized T-cells. Our data show that LPS had no significant effect on the expression of MHC class II molecules on the surface of mammary macrophages. Furthermore, LPS-induced macrophages were no more active in supporting T-cell proliferation on a per cell basis than unstimulated macrophages. The lack of macrophage response to LPS with respect to expression of MHC class II molecules and the antigen presentation ability is another example of the hyporesponsive nature of macrophages isolated from the bovine mammary gland.     

牛皮下注射爱普菌素的组织药代动力学试验

牛皮下单次注射爱普菌素注射剂,剂量为0.5 mg/kg,给药后在不同时间点采取肌肉、肝脏、肾脏和脂肪等组织样品检测爱普菌素残留,采用3P97软件对组织残留-停药时间数据进行分析.结果表明,注射部位、肝脏中爱普菌素残留浓度变化符合二室开放模型,肌肉、肾脏、脂肪中EPR残留浓度变化符合一室开放模型.爱普菌素经皮下注射后Tmax均小于1 d(0.17～0.76 d),Cmax范围在37.32～1453.79 ng/g之间.MRT范围在7.54～14.79 d之间,与T1/2el范围2.91～19.50 d相一致,说明药物在动物体内消除缓慢.     

Foot ash can replace tibia ash as a quantification method for bone mineralization in broilers at 21 and 42 days of age

Prior research indicated that foot ash determinations were as robust as tibia bone ash determinations in reflecting the degree of bone mineralization in chicks at 14 d of age. In the current research, the relative effectiveness of the 2 procedures was evaluated in 21- and 42-day-old broilers while also evaluating a new dietary phytase supplement. In experiment 1, broilers were fed until 21 d of age a negative control diet with 0.24% available phosphorus, a positive control diet with 0.48% available phosphorus, or the negative control diet supplemented with 300, 500, 1,000, or 2,500 phytase units/kg diet. In experiment 2, broilers were fed until 42 d of age negative control diets having 0.275, 0.250, and 0.225 percent available dietary phosphorus in the starter, grower, and finisher periods, respectively, positive control diets having 0.475, 0.450, and 0.425 percent available dietary phosphorus in the starter, grower, and finisher periods, respectively, or the negative control diets supplemented with 500, 1,000, or 2,500 phytase units/kg diet. At 21 and 42 d of age, broilers fed diets supplemented with the 2 highest doses of phytase had foot and tibia ash values equal to those fed the positive control diet and higher than those fed the negative control diet. At 42 d of age, feed conversion and total breast meat yield values for the broilers fed the highest dose of phytase were superior to the values of the birds fed the positive control diet or the diet containing the lowest dose of phytase. The results indicate that adding levels of this new dietary phytase beyond what is necessary for normal bone mineralization enhances feed conversion and that dried foot and tibia bone ash determinations are both reliable in detecting differences in bone mineralization in 21- and 42-day-old broilers.     

Quantification of mammary gland tissue size and composition changes after weaning in sows

The objectives of this study were to characterize the tissue compositional changes in porcine mammary glands after weaning and to determine whether administration of estradiol alters the profile of these tissue changes. Forty-five primiparous sows were assigned randomly to one of two treatment groups after weaning, control or estrogen treated. Estrogen-treated sows received twice-daily injections of estradiol-17beta (0.125 mg/kg of BW); control sows received vehicle injections. Sows were weaned at d 21 of lactation and killed on either d 0 (d of weaning; n = 5) or on d 2, 3, 4, 5, or 7 after weaning (n = 4 per treatment on each day). Teat order relative to suckling behavior was observed on the day before weaning to determine which mammary glands the piglets suckled. Suckled and non-suckled glands were identified from the teat order observation, and individual mammary glands were collected at slaughter. Mammary glands were trimmed of skin and extraneous fat pad, individually weighed, and bisected to measure cross-sectional area. The remaining half of each gland was ground and stored at -20 degrees C for chemical analyses. Frozen tissue was used for measuring tissue DNA, DM, protein, fat, and ash contents. Suckled mammary glands of sows undergo significant and dramatic changes during the initial 7 d after weaning, with significant changes occurring even by d 2 after weaning. Mean cross-sectional area of parenchymal tissue in suckled mammary glands decreased from 59.7 +/- 2.1 cm2 on the day of weaning to 26.8 +/- 2.3 cm2 by d 7 after weaning (P < 0.0001). Mammary gland wet weight decreased from 485.9 +/- 22.0 g on the day of weaning to 151.5 +/- 24.8 g by d 7 after weaning (P < 0.0001), whereas DNA decreased from 838.8 +/- 46.2 g on the day of weaning to 278.4 +/- 52.5 g by d 7 after weaning (P < 0.0001). The changes in gland wet weight and DNA during the period of mammary gland involution in the sow represent loses of over two-thirds of the parenchymal mass and nearly two-thirds of the cells that were present on the day of weaning. Estrogen treatment did not affect overall mammary involution during the first 7 d after weaning. Mammary glands that were not suckled during lactation had no further loss of parenchymal tissue during the first 7 d after weaning. Mammary gland involution in the sow is a rapid process and is probably irreversible within 2 or 3 d after weaning.     

Effect of bovine growth hormone on incorporation of [14C]acetate into lipids by co-cultures of bovine mammary, liver, and adipose tissue explants

Incorporation of [14C]acetate into lipids was measured in 24 hr co-cultures of mammary, liver and adipose tissue from Holstein cows at 53, 210 and 318 d of lactation in the presence or absence of bovine growth hormone. Little (less than 1%) of the labeled lipids appeared in the media relative to that incorporated into the tissue. In mammary tissue, incorporation of [14C]acetate was highest into triglycerides (16,298 cpm/mg mammary tissue), followed by phospholipids (1,887 cpm), free fatty acids (1,252 cpm), diglycerides (708 cpm), free cholesterol (360 cpm) and monoglycerides (93 cpm). Bovine growth hormone did not increase incorporation of [14C]acetate when mammary or adipose tissue were incubated separately. However, in the presence of liver and adipose tissue, bovine growth hormone significantly increased the incorporation of [14C]acetate into triglycerides, diglycerides, free fatty acids and free cholesterol by mammary tissue. These results suggest that bovine growth hormone acts on mammary tissue indirectly through liver and adipose tissue to increase lipid synthesis. This mechanism may play a role in the action of bovine growth hormone in vivo to increase milk and milk fat production.